Many parameters of the helper T cell response are decreased in the aged. Aged murine Th cells show reduced mitogen- and antigen-induced proliferation and IL-2 production and diminished help for B cells and CTL. In contrast, IL-4 production is increased in aged T cells. This suggests that these defects may result from a change in the relative proportion of different subpopulations of Th cells alterations in activation and intracellular signalling events that occur in CD4+ T cells with aging will first be carefully defined in 3 mouse strains. Proliferation, lymphokine production (IL-2, IL-4, IL-5, ifn-gamma), increases in cytoplasmic calcium concentration and PIP2 hydrolysis will be examined in response to different stimuli. CD4+T cell responsiveness to cytokines and accessory cell function will also be studied in this initial definition of the system. Second, alterations in Th subpopulations differing in lymphokine production (Th1, Th2 and ThO cells) will be directly evaluated in cells freshly isolated from young and old mice. CD4+ T cells will be activated in vitro and lymphokine production profiles will be measured using three different single cell assays: 1) in situ hybridization; 2) ELIspots for secreted lymphokine; 3) limiting dilution cloning directly out of the animal followed by detection of lymphokine MRNA by PCR. Third, to determine whether changes in previously defined Th subpopulations can explain the functional defects observed with aging, CD4+ T cells from young and old mice will be separated by flow cytometry based on the magnitude of the calcium signal generated upon activation, and on differential expression of FcRmu, Pgp-1, CD45RB or IL-1R. The sorted populations will be studied to determine relative proportions of Th1, Th2, and ThO cells and cytokine responsiveness. Flow cytometry will also be used to determine surface marker expression of cells differing in the strength of the calcium signal, and cross-correlation of expression of the markers listed above. Fourth, it will be determined whether antigen-specific responses behave comparably to polyclonal responses. Old and young mice will be primed with the antigens KLH, gp160 428-443 and a crude extract of Leishmania major promastigotes. Following in vitro restimulation with antigen, proliferation and lymphokine production patterns will be examined. The studies proposed in this grant application should greatly enhance our understanding of the alterations in Th cell function that occur with aging.